The main sources of hematopoietic stem cells (HSCs) are the bone marrow and the umbilical cord blood (UCB). HSCs are used in the transplantation setting (autologous or allogeneic) which constitutes one of the most effective treatment strategies for achieving cures in patients with hematologic malignancies, bone marrow failure conditions, a variety of congenital diseases of global concern (e.g. sickle cell anemia and thalassemia) and autoimmune diseases such as lupus. However, this opportunity for life-saving or life-improving treatment is not available to many thousands of people worldwide due to an inability to amplify these cells ex vivo sufficiently to make the procedure safe and successful. More particularly, for every 3 patients, one will forego the opportunity for transplant because no human leucocyte antigen (HLA) identical donor can be found. Another proportion of patients will not have access to transplantation simply because too few HSCs are available in the graft (i.e. cord blood or autologous) for successful transplant. The safety and efficacy of marrow transplant is directly dependent on the number of HSCs and progenitor cells available for engrafting. The more that can be infused, the more rapidly is hematologic function restored, and the shorter is the window of risk for infection due to lack of granulocytes or of bleeding due to lack of platelets. The challenge in providing sufficient HSCs is further escalated where non-myeloablative conditioning is preferred such as in the context of gene therapy for major inherited blood disorders (the major genetic cause of morbidity and mortality worldwide).
In adults, HSCs mainly reside in the bone marrow and must be mobilized to enter the circulation prior to being collected by apheresis, either for autologous or allogeneic hematopoietic stem cell transplantation (HSCT). The collection of an adequate number of CD34+ cells, a surrogate marker of (HSCs), is paramount because the dose of CD34+ cells influences the success and rate of hematopoietic recovery. Several reports suggest that a higher infused CD34+ cell dose is independently predictive of improved survival.
The two most commonly used mobilizing regimens are granulocyte-colony stimulating factor (G-CSF) and G-CSF plus chemotherapy. Plerixafor, a CXCR4 antagonist approved by the United States Food and Drug Administration (FDA) in 2008 and in 2011 by Health Canada, enhances mobilization of HSCs when administered with G-CSF. However, Plerixafor is contraindicated in patients with leukemia because of mobilization of leukemic cells. Inability to obtain sufficient numbers of CD34+ cells/kg with currently used mobilization regimens is estimated to affect up to 15% of patients (varies between diseases). Use of autologous HSCT in hematological malignancies is often limited by the fact that both normal and cancer stem cells are present in the bone marrow and thus, likely to be mobilized.
Allogeneic HSCT with BM or mPBSC is another transplantation alternative. However, about one third to one fourth of the patients who are eligible for this type of transplant cannot find a suitable donor. For those who get transplanted, there is a high frequency of transplant related mortality due to graft-versus-host disease, relapse or graft rejection; and a risk of immunodeficiency for prolonged periods of time. Alternatively, umbilical cord blood has been shown as a valid option in allogeneic HSCT. However, a single CB unit typically provides insufficient HSCs for an adult patient for a rapid and efficient hematopoietic recovery.
In vitro conditions that support cytokine-mediated short-term maintenance or even modest increase in murine or human HSC numbers measured by mouse reconstitution assays are generally accompanied by much more robust increase in later types of progenitor cell populations. More marked increases in murine and human HSCs have more recently been described in cultures containing other factors such as fibroblast growth factor (FGF), insulin-like growth factor binding proteins, angiopoietin-like growth factors and pleiotrophin. However, these latter reports are thus far solitary and await independent confirmation. Short-term increases in HSCs obtained with standard cytokines in vitro are also inevitably followed by eventual HSC depletion.
Alternative strategies for human HSC expansion have involved their culture with stromal elements or soluble morphogenic ligands (e.g. stimulating the Notch, Wnt and Hedgehog pathways), targeted manipulation of specific intracellular signaling pathways (PGE2, ROS, p38 and MAPK inhibitors) or manipulation of specific transcription factors (e.g. Hox, HIf). Other preclinical approaches for ex vivo expansion of HSCs include incubation with: i) StemRegenin1 (SR1), an aryl hydrocarbon receptor antagonist (Boitano, A E et al. “Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells” Science 329: 1345-1348. 2010); ii) Garcinol, a histone acetyltransferase inhibitor (Nishino, T et al. “Ex vivo expansion of human hematopoietic stem cells by Garcinol, a potent inhibitor of histone acetyltransferase” PLoS ONE 6(9): e24298. 2011); and iii) NR-101, a non peptidyl small molecule c-MPL agonist (Nishino et al. “Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL” Exp. Hem. 2009; 37:1364-1377). Characterization of SR1 provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion.
Clinical studies have stressed the requirement not only for permanence of the administered transplants, but also the importance of minimizing the time to appearance of useful granulocyte levels post-transplant which, in turn, depends on the number of short term repopulating cells infused. Transplantation of marrow or cord blood cells expanded in culture with cytokines has not so far demonstrated clinically useful acceleration of hematopoietic recovery compared to untreated cells. Early results of trials with cells expanded using immobilized Notch ligands have been the first to show potential clinical utility for any (even modest) progenitor cell expansion strategy (Delaney et al. “Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution” Nat. Med. 16(2): 232-236. 2010). This approach is however limited by the need to use an immobilized Delta-1 fusion protein during the ex vivo expansion step and by the lack of documented effect on stem cells (impact appears limited to more differentiated progenitors). Other approaches in clinical trial include: i) StemEx, a combination of UCB cells cultured with the copper chelator tetraethylenepentamine (TEPA) and cytokines, co-infused with non-treated UCB cells; phase I results show that time to neutrophil or platelet engraftment was not improved compared to previous reports (de Lima M et al. “Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial” Bone Marrow Transplant. 2008; 41(9): 771-778); and 16-16 dimethyl prostaglandin E2 (PGE2), used for improving homing of UCBT in a phase I trial.
There is thus a need for novel strategies for increasing the expansion of hematopoietic stem and progenitor cells. Certain pyrimido[4,5-b]indole derivatives are known in the art that are used in that regard; they are disclosed for example in: WO 2003/037898; WO 2004/058764; WO 1998/042708; WO 1997/002266; WO 2000/066585; WO 1993/020078; WO 2006/116733; WO 2008/055233; WO 2010/006032; WO 1995/019970; WO 2005/037825; and WO 2009/004329. However, these documents do not disclose the pyrimido[4,5-b]indole derivatives according to the invention or their use in the expansion of hematopoietic stem and progenitor cells.